Pap staining is used to differentiate cells in smear preparations of various bodily secretions; the specimens can be gynecological smears (Pap smears), sputum, brushings, washings, urine, cerebrospinal fluid, abdominal fluid, pleural fluid, synovial fluid, seminal fluid, fine needle aspiration material, tumor touch samples, or other materials containing cells.
The classic form of Pap stain involves five dyes in three solutions:
When performed properly, the stained specimen should display hues from the entire spectrum: red, orange, yellow, green, blue, and violet. The chromatin patterns are well visible, the cells from borderline lesions are easier to interpret, the photomicrographs are better, and the stained cells are pretty. The staining results in very transparent cells, so even thicker specimens with overlapping cells can be interpreted.
On a well prepared specimen, the cell nuclei are crisp blue to black. Cells with high content of keratin are yellow, glycogen stains yellow as well. Superficial cells are orange to pink, and intermediate and parabasal cells are turquoise green to blue. Metaplastic cells often stain both green and pink and once.
Pap stain is not fully standardized; it comes in several versions, subtly differing in the exact dyes used, their ratios, and timing of the process.
The EA stain contains two mutually incompatible chemicals, Bismarck brown and phosphotungstic acid, which precipitate each other, impairing the useful life of the mixture and compromising the differential staining of eosin and light green. The descriptions of the compositions of the staining solutions vary by source and differ even in Papanicolaou's own publications. Mixtures of the same name from different vendors therefore can differ in composition, occasionally producing different or poor results.
Ultrafast Papanicolaou stain is an alternative for the fine needle aspiration samples, developed to achieve comparable visual clarity in significantly shorter time. The process differs in rehydration of the air-dried smear with saline, use 4% formaldehyde in 65% ethanol fixative, and use of Richard-Allan Hematoxylin-2 and Cyto-Stain, resulting in a 90-second process yielding transparent polychromatic stains.